Investigating Srsf3-mediated alternative RNA splicing in craniofacial development

项目摘要 颅面发育涉及复杂的信号协调组织组织形成头部 和脸，在这个过程中的中断导致常见的先天性畸形。这其中的一个关键问题 外部刺激如何导致形成完全发育的颅面所需的基因表达变化 结构.通过血小板源性生长因子受体α（PDGFRα）的信号传导在以下方面起着关键作用： 这一过程，因为PDGFR A中的突变与人类的唇腭裂相关。相关地，Pdgfra突变体 小鼠模型产生了从腭裂到完全面部裂开的一系列表型。酰肌醇 3-激酶（PI 3 K）是小鼠骨骼发育过程中PDGFRα信号传导的主要效应子， Akt激酶的激活。先前的磷酸化蛋白质组学筛选表明Akt磷酸化 RNA结合蛋白（RBP）富含丝氨酸/丝氨酸的剪接因子3（Srsf 3）在PI 3 K介导的 小鼠胚胎腭间充质（MEPM）细胞中的PDGFRα信号转导，导致PDGFR α易位。 磷酸化Srsf 3进入细胞核。Srsf 3在神经系统中广泛表达， 在妊娠中期，小鼠面部突起的嵴源性间充质和上覆的外胚层。此外，本发明还 在鼠神经嵴细胞谱系（cKO）中的Srsf 3的消融导致严重的中线面裂 由于颅神经嵴细胞的增殖和存活缺陷而导致的表型。此外，RNA测序的 Srsf 3 cKO面突间充质鉴定了选择性RNA剪接事件，其富集了 转录编码蛋白丝氨酸/苏氨酸激酶，这表明选择性剪接可能作为一种新的 细胞内激酶信号传导的反馈机制。本提案的目的是检验以下假设： PI 3 K/Akt介导的PDGFRα信号调节Srsf 3蛋白和RNA相互作用以影响选择性 颅面发育所必需的转录本的RNA剪接。首先，Srsf 3将从 MEPM细胞在PDGF-AA配体不存在或存在的情况下，并通过质谱分析， 全面绘制PDGFRα信号转导引起的磷酸化变化。此外，颅面 将在Srsf 3磷酸突变体敲入小鼠模型中分析表型，以确定Akt的作用。 Srsf 3在颅面发育中介导的磷酸化。接下来，BioID 2邻近标记和质量分析。 光谱法将用于鉴定Srsf 3蛋白相互作用伴侣，以响应PDGFRα信号传导， MEPM细胞。最后，将纯化Srsf 3-RNA相互作用并测序以响应PDGFRα信号传导 在MEPM细胞中通过增强的交联和免疫沉淀分析来鉴定 Srsf 3，并确定RNA结合和/或序列特异性是否在Srsf 3磷酸化后发生变化。这 该项目将确定Srsf 3活性响应PDGFRa的分子机制 信号在面部间充质，从而提供了相当深入的机制，基因 在哺乳动物颅面发育过程中的表达调控。

Project Summary Craniofacial development involves complex signaling to coordinate tissue organization to form the head and face, and disruptions in this process result in common congenital malformations. A key question in this field is how external stimuli lead to gene expression changes required to form fully developed craniofacial structures. Signaling through the Platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in this process, as mutations in PDGFRΑ are associated with cleft lip/palate in humans. Relatedly, Pdgfra mutant mouse models develop a range of phenotypes from cleft palate to complete facial clefting. Phosphatidylinositol 3-kinase (PI3K) is the primary effector of PDGFRα signaling during skeletal development in the mouse, leading to the activation of the kinase Akt. A previous phosphoproteomic screen demonstrated that Akt phosphorylates the RNA-binding protein (RBP) Serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to translocation of phosphorylated Srsf3 into the nucleus. Srsf3 is ubiquitously expressed with enhanced expression in the neural crest-derived mesenchyme and overlying ectoderm of mouse facial processes at mid-gestation. Additionally, ablation of Srsf3 in the murine neural crest cell lineage (cKO) results in a severe midline facial clefting phenotype due to defects in proliferation and survival of cranial neural crest cells. Further, RNA-sequencing of Srsf3 cKO facial process mesenchyme identified alternative RNA splicing events that were enriched for transcripts encoding protein serine/threonine kinases, suggesting that alternative splicing may serve as a novel feedback mechanism for intracellular kinase signaling. The goal of this proposal is to test the hypothesis that PI3K/Akt-mediated PDGFRα signaling regulates Srsf3 protein and RNA interactions to affect the alternative RNA splicing of transcripts necessary for craniofacial development. First, Srsf3 will be immunoprecipitated from MEPM cells in the absence or presence of PDGF-AA ligand and analyzed by mass spectrometry to comprehensively map phosphorylation changes in response to PDGFRα signaling. Further, craniofacial phenotypes will be analyzed in a Srsf3 phosphomutant knock-in mouse model to determine the role of Akt- mediated phosphorylation of Srsf3 in craniofacial development. Next, BioID2 proximity labeling and mass spectrometry will be used to identify Srsf3 protein interacting partners in response to PDGFRα signaling in MEPM cells. Finally, Srsf3-RNA interactions will be purified and sequenced in response to PDGFRα signaling in MEPM cells through enhanced crosslinking and immunoprecipitation analysis to identify direct targets of Srsf3 and determine if RNA binding and/or sequence specificity changes upon Srsf3 phosphorylation. This project will determine the molecular mechanisms by which Srsf3 activity is controlled in response to PDGFRa signaling in the facial mesenchyme, thus providing considerable insight into mechanisms underlying gene expression regulation during mammalian craniofacial development.